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2010 Volume 2 Issue 4

Photoaffinity labeling of Aminoglycoside Phosphotransferase 3’-II with azido ATP confirms the involvement of conserved lysine in ATP binding


Abstract
Of the mechanisms bacteria use to evade the effects of aminoglycoside (AG) antibiotics, enzymatic modification has the most clinical relevance due to the promiscuous nature of the DNA encoding the genes for these enzymes. One such enzyme, aminoglycoside 3’-phosphotransferase IIa (APH(3’)-IIa), is used as a model for understanding this modification at a molecular level along with anticipating the evolution of AG resistance. To study the structure-function relationships of this enzyme, we previously determined the crystal structure of APH(3’)-IIa and modeled its ATP binding site. We identified a lysine that appeared to be involved in the binding of ATP and generated a conserved lysine to arginine mutant to better assess this residue’s functional contributions. We determined the binding kinetics for ATP to this mutant and employed active-site labeling using an azido ATP analog to confirm the importance of this conserved lysine. Competition experiments with tryptophan during activesite labeling revealed that a conserved tryptophan residue in the amino-terminus of the enzyme may be involved directly or by association with ATP binding. Together, these data give us a clearer picture of how ATP associates with APH(3’)-II and could aid in the development of small chemical compounds which specifically inhibit its activity.

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J BIOCHEM TECH
J BIOCHEM TECH
Journal of Biochemical Technology is a peer reviewed International Journal published by the Sevas Publishing on behalf of the Biochemical Technology Society, a Registered Charity Organization from India

AREA OF INTEREST
AREA OF INTEREST
Areas of high interest, cover new advances in enzymatic and protein mechanims; applied molecular genetics and biotechnology; genomics and proteomics; metabolic; medical, environmental, food and agro biotechnology.

FOCUS AND SCOPE
FOCUS AND SCOPE
Journal Of Biochemical Technology” Provides A Medium For The Rapid Publication Of Full-Length Articles, Mini-Reviews Of New And Emerging Products And Short Communications On All Aspects Of ...

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This journal provides immediate open access to its content on the principle that making research freely available to the public supports a greater global exchange of knowledge. Keywords include, Biochemical Research: Endo/exocytosis, Trafficking, Membrane Biology, Cell Migration, Cell-Matrix Organelle Biogenesis, Cytoskeleton Proteolysis, Cell Death, Cell Cycle, Cancer, Cell Growth/Death, Differentiation, Drug Targets, Gene Therapy, Models of Disease, Proteomics, Stem Cells, Bioenergetics, Mitochondria, Free Radicals, Redox Signaling, Ion Transport/Channels, Oxidative